I want to ask, for how many time (years), a bacterial culture remain preserved with 40% glycerol at -80C ?. In the other sense if a culture remain in glycerol for 2 years then it have any effect on the efficiency and activeness of strain or need to refresh culture after every 6 months?.
Looks like I am the dinosaur here when I tell you that my glycerin stocks from 1992 (> 20 years) are still absolutely OK (E.coli). They even survived several moves from one country to another in my own car, where they thawed of course (from Germany to Switzerland, back to Germany and finally to France).
I did MLST genotyping on bacteria that were stocked in 20% glycerol under -80C since 7 years ..... their growth was good and strong and genetically stable.
I think the known condition that changes bacterial characteristics (specially pathogenecity and antibiotic resistance) is several subculture of the same colonies
If it is for particular period of time (2 to 3 years), atleast temperature that is lesser than -30 degree C may be upto -80 degree C the culture remains well active and we can see the revival and efficiency too.
1. I use 15-20% glycerol (autoclaved). It can stay at -80oC for at least several years. I would also store the plasmid (use to transforme the bacteria) at a safe place in case you need it again.
2. Store several tubes if space allowed; no need to thaw the culture to get the bacterial colonies
3. Re-transform with the same plasmid is preferred than to 're-store' (by subculturing) the same clones after a period time of use
It depends up on type of the culture you are handling. and this varies from strain to strain. with endospore forming bacteria you can revive this after 2 to 5 years provided, the culture has not undergone any freezing-thawing cycle.
Check, whether the preserved culture is solidified or not at -80 degree. When i was in MTCC, IMTECH, India i used to preserve any bacterial culture using sterile 10% glycerol (v/v). But you can use 20%. Please remember, while you are reviving you must not thaw it. you can use sterile tooth pick (wooden) to transfer small bit of solidified 10% glycerol stock and replace back in the respective slot (box, to be stored at -80) as soon as possible.
You must check viability of culture at least every once in three months on solid agar plate.
I think its going to be depending of kind of organisms, in my case culture of overnight samples of enterobacterial ambiental strains with 50% of glycerol is a good method, without important changes at least by 5 years, efficiency 80% aproximately, modified strains from ATCC are very unstable, one year with the same properties according to some ISO methods
Is your final concentration of glycerol 40%? If so that is a little high.15-25% v/v works best. They should be fine for years. I've used bacterial freezer stocks from about 8 years ago.
i am dealing with 40% glycerol and under studied all strains belong to Pseudomonas group
Looks like I am the dinosaur here when I tell you that my glycerin stocks from 1992 (> 20 years) are still absolutely OK (E.coli). They even survived several moves from one country to another in my own car, where they thawed of course (from Germany to Switzerland, back to Germany and finally to France).
Is it important for you to use 40% glycerol, why not the standard15-25% solution?
I made similar experience as Ralf, I still use glycerol stocks from my PhD thesis and the stocks are more than 20 years old, but I never thawed them (transport on dry ice), always scraped a little bit substances from the surface with a sterile pipette tip.
In my experience, glycerol stocks (40% or 25% v/v) at -80C can be preserved by decades. The efficiency and activeness of a bacterial strain should´t decrease with time.
In my experience those stocks, depending on the bacterial species preserved, can last for no more than 5 years. Same thing for DMSO as a cryoprotectant. We have seen strains loosing valuable plasmids.
I have cultures in 8% glycerol stored in -80C that are still excellent after 5 years.
I made good experience keeoing glyceol cultures in a mixture of 1% Bacto Peptone salt free mixed with equal volumes of 80% glycerol. Salt free is important. I have still cultures growing which date about 30 years ago stored at -80°C. Important if you take samples from them never thaw them but just go with you loop on the surface and inoculate from ice. Temperatures above -80% might be critical in peculiar -20 as the frigerator often works in cycles perfoming "freezing and thawing" the structure in the culture can rapidly change.
Same response as all the others. I have bacterial stock kept in -80 for the last 3 years. I can still innoculate and they grow. I even use 25% glycerol and no issues. Just be careful not to freeze thaw.
prepare 80% glycerol and store at 1:1 ratio (i.e. 500µl of 80% glycerol and 500µl of culture). sterilize the 80% glycerol by autclaving and use distilled water for preparation.
I think bacterial culture in 40% glycerol will be stable for years at -80oC.
I stored my cultures in -20 C for more than one year and it stills vial and active... so in your case (-80) it would be more years.
Your question isnt clear ,is it transformed bacteria or just ordinary strain under storage. If transformed then you need to confirm presence of introgress when using it after lengthy storage otherwise as long as the glycerol preservative contains the necessary support media to keep cells nourished the glycerol will protect against freezing which damages the cells - can store for long ,l have worked with bacteria (mycobacteria ,e coli, agrobacteria) which have been in storage for more than 5 years without change but some transformed E coli did lose their transformation property.
The glycerol stocks of bacteria remain intact in their genetic characteristics, compared to frequently sub-cultured strains.
I agree with other Scientist people in preserving bacteria in glycerol for many years without destruction.
I'm working in a culture collection center and we have stocks of Bacteria in 10-15% glycerol combined with other suspending media such as 15-20% skim-milk in -80 C which are remained intact for more than 30 years and will revive in good conditions. I'm agree that sub-culturing will reduce the parthenogenesis and some other characteristics of the organism specially when the gene is located on the plasmids.
What concentration of Glycerol is optimum ? or toxic? This is the question. Also what media is good? TSA with 15% glycerol I use and heard that more is toxic Is it true? Anybody Knows?
Dear Gholam, The optimum concentration is 15 to 40% sterile glycerol to bacterial culture but ideal is 15 to 20% for all media. Anything over 10% should be ok. The point of the glycerol is prevent freezing damage, such as ice crystal formation.
At lower glycerol concentrations like 10-20% the frozen stock will be fairly solid at -80C, while at higher concentrations (30-50%) it may remain partially liquid. Either is fine but over time (years) the cells will settle down at bottom if still liquid.
As suggested by all, to recover the bacteria, don’t thaw the vial but simply scrape the frozen surface of the culture and seed in plate or medium.
Dear Muhammad Naveed,
We mix Glycerol in medium while storing bacterial cells and store them @ -80ᵒC. Actually, Glycerol is a non-ionic kosmotrope (co-solvent). It forms strong, stable and durable hydrogen bonds with water molecules in the medium. In this way, it competes water-water hydrogen bond formation and disrupts the crystal lattice formation and prevents our cells from crystal mediated destruction. Moreover, Glycerol is lighter than water or medium. So, when we add Glycerol in medium for storage purpose what exactly happens is that Glycerol makes a layer on the upper meniscus of the medium containing bacterial cells. Thus, this layer of Glycerol prevents harmful water crystal mediated destruction of our cells from outside the layer. Well, some lipophilic bacteria may utilize this glycerol while becoming active from a long dormant stage.
Hope you find it useful.
Good luck
Regards
Dear Gholam Reza Talei
Addition of Glycerol in medium for storage purpose depends on several aspects. Well, in bacterial strain storage @ -80ᵒC, we add 10% or more Glycerol. Although, in semisolid BHA (Brain Heart Infusion Agar) for bacterial strain storage, 1% Glycerol is also suggested, but that must be stored @ refrigerator not @ -20ᵒC or -80ᵒC.
Good Luck
Regards
Thanks Surajit Chakraborty for explaining a comprehensive mechanism of glycerol storage.
i isolate slight and moderate and extreme halophilic bacteria .. i want to preserve it in glycerol for 1 year i made broth culture of its isolation medium then take 500 microlitre on 500 microlitre of (25%) glycerol then freezing ... is this true or not ??
My experience is good with E. coli but not so good with Vibrio vulnificus. In some cases isolates of V. vulnificus lost their viability after months stored in glycerol, so I guess it's best if you have a back up storing alternative
Hi Heba,
Yup, it is all right, you can preserved it up to one year in this conditions.
Just maintain cold conditions like -20C or for better at -80C and glycerol 20% to 40% is ideal for this regards.
stay happy
I think if any bacterial culture subculture again and again so must be lost it's own efficiency.
There are few things that you should keep in mind while keeping/storing/preserving a bacterial culture at -80. First ensure that for your culture this method works well. Then go ahead for preservation. Now prepre at least three replicates (in three cryo vials, 0.5 to 0.75 ml eac) for each culture. take a viable count (cfu/ml) of the culture that your preserving. Normally you go for a very dense culture with viable count of about 10 to the ppower 10 or 11 /ml or even more. In myexperiance, if there is no power failure, if there is no freezing thawing, your cultures should remain viable for 3 to 5 years. I got this during my stay at MTCC, Chandigarh, India. It woks well for all bacterial cultures (heterotrophs). The glycerol % that we used was 10%. Please ensure that preservation is carried out properly, following proper methodology. You should have proper inventory so that you do not spend time while reviving your culture. Nor mally you should check viability of your culture once in a year. you would see there will be decrease from 10 to 100 fold. so that make sence if you have a very dense starting culturing. Hope it helps.
Best Wishes
150
Hi Muhammad
You can find some useful references on attachment 1
https://www.opsdiagnostics.com/notes/ranpri/aguidetobacteriapreservation.htm
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2
https://www.researchgate.net/file.PostFileLoader.html?id=58a09071b0366dac2449c49d&assetKey=AS%3A460980590256128%401486917745083
4
https://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822010000400023
5
https://www.scielo.br/scielo.php?script=sci_arttext&pid=S1806-66902013000400001
7
https://www.rapidmicrobiology.com/test-method/stock-culture-maintenance-and-storage/
8
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=10&cad=rja&uact=8&ved=0ahUKEwiYjb-N94HUAhUQY1AKHd9OBhsQFghoMAk&url=https%3A%2F%2Fwww.cedarlanelabs.com%2FContents%2FFiles%3FfilePath%3Dpdf%255CATCC%2520-%2520Bacterial%2520Culture%2520Guide.pdf&usg=AFQjCNGHxQ_6IQkkMFAv2J6U5Dq9yWFQLw&sig2=jtNSK7aB5Vz5olM3C14nZA
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https://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=3698&context=rtd
10
https://pubmedcentralcanada.ca/pmcc/articles/PMC187002/pdf/applmicro00022-0044.pdf
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https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=16&cad=rja&uact=8&ved=0ahUKEwiar7Gx-IHUAhVOblAKHeW0AHM4ChAWCEYwBQ&url=http%3A%2F%2Fmic.microbiologyresearch.org%2Fcontent%2Fjournal%2Fmicro%2F10.1099%2F00221287-26-3-367%3Fcrawler%3Dtrue%26mimetype%3Dapplication%2Fpdf&usg=AFQjCNHPhToY1eWhorrsG8U2x7bHVj4KhA&sig2=sxfx8-wvvhIpw-eYTAIzVQ
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https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/Brothswith15Glycerol.htm
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